In this study, for the first time, in vivo, TLR4 and SYK were colocalized and had a high correlation in mastitis mammary epithelium; protein–protein interaction results also confirmed that there was a direct interaction between them in mastitis tissue, suggesting that SYK participates in the immune regulation of the TLR4 cascade for bovine mastitis. The gene discussed is SYK; the disease is mastitis.